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American Laboratory

Practical Resources for Laboratory Scientists

February 2012

Volume 44, Number 2 Attend Pittcon 2012 to Connect With Colleagues, Innovations, and Scientic Discoveries Fast and Effective LC/MS/MS Analysis of Synthetic Cannabinoids Purchasing Considerations for Field Soil Water Content Prole Probes Automated Powder Dosing in the Life Science Laboratory Food Safety Testing and New Technologies Pesticide Residue Testing of Grains and Oil Seeds: Minimizing False Positives and False Negatives Optimizing HPLC Sample Preparation Utilizing Extended Linear Velocity to Maximize Peak Capacity in UHPLC Do Your Work Cleanly With Gloveboxes Highlights and Challenges in Laboratory Automation: Review of the 4th Annual Automation User Group Meeting Statistics in Analytical Chemistry Part 46R2 (Concluded)


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American Laboratory
Volume 44, Number 2

Life Science
Automated Powder Dosing in the Life Science Laboratory by J. Prochnow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pg 16
As demonstrated in this article, automated and gravimetric sample and standard preparation signicantly reduces the cost of compliance, making the process more reproducible and improving the analytical results.

Food and Beverage

Food Safety Testing and New Technologies by M.M. Shukla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pg 22
New food technologies are revolutionizing the food safety testing industry.

Pesticide Residue Testing of Grains and Oil Seeds: Minimizing False Positives and False Negatives by R. Trengove, B. Peebles, K. Rousetty, S. Bong, F. Muntean, S. Schachterle, C. Kellog, and P. Jeaville . . . . . . . . . . . . . . . . . . . . . . . . . pg 25
The matrix can signicantly affect the GC-MS analysis of certain pesticides, as demonstrated in the study described in this article.

What Is This?
This months cover image was provided by Dr. Kavita Aswani of Lumen Dynamics, Mississauga, Canada. The image was captured using an X-Cite XLED1 uorescence microscopy illumination system; original magnication 20. Fluorescent microscopes and uorescence imaging systems are useful laboratory instruments that share similar methods to visualize specimen and sample images using uorescence. What do you think it is? To comment, go to See the answer to the January cover on page 48.

Optimizing HPLC Sample Preparation by C. Smith . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pg 32
Sample preparation techniques for high-performance liquid chromatography (HPLC) and ultra high-performance liquid chromatography (UHPLC) merit careful consideration.

Fast and Effective LC/MS/MS Analysis of Synthetic Cannabinoids by E. Pike, M. Rummel, M. Trass, J. Layne, and S. Countryman . . . . . . . . pg 9
Toxicology labs rely on LC/MS/MS to provide sensitive analysis of synthetic cannabinoids.

Utilizing Extended Linear Velocity to Maximize Peak Capacity in UHPLC by D. Stickle and B. Giuffre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pg 38
Increasing ow rate, gradient time, and column temperature resulted in an increase in the peak capacity of the column lengths studied.

Product Intelligence
Do Your Work Cleanly With Gloveboxes by J. Netterwald . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pg 36

Purchasing Considerations for Field Soil Water Content Prole Probes by E.S. Tozzi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pg 13
Prospective purchasers of probes for soil water content prole must carefully consider their capabilities and limitations.

Conference Review
Highlights and Challenges in Laboratory Automation: Review of the 4th Annual Automation User Group Meeting by R.L. Stevenson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pg 42


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Statistics in Analytical Chemistry Part 46R2 (Concluded) by D. Coleman and L. Vanatta. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pg 44

Call for Cover Images

American Laboratory is now opening its doors for cover image submissions. Become a part of our 40-year history of vibrant, thought provoking covers. Showcase your imaging and microscopy techniques.

New Products
Product Comparison: Water Baths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pg 41 Product Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pg 47 Laboratory Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pg 47

Editors Page Attend Pittcon 2012 to Connect With Colleagues, Innovations, and Scientic Discoveries by J.N. Peace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pg 6 Advertising Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pg 48

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American Laboratory (ISSN 0044-7749) Volume 44, Number 2 Free subscriptions are available to qualied individuals. To request, edit, or cancel a subscription to American Laboratory, please visit Published 10 times per yearmonthly except June/July and November/December when combined by American Laboratory/Labcompare, 30 Controls Drive, Shelton, CT 06484-0870, U.S.A; Tel.: 203-926-9300; Fax: 203-926-9310; e-mail: info@; Home Page: Periodicals postage paid at Shelton, CT, and additional mailing ofces. Printed subscriptions available, 2012 subscription rates: $400 in continental U.S.A.; $1075 in other countries (including postage, surface rate). Digital subscriptions available: $275. Call for agency discount. Back issues: $60.00 per issue. Connecticut residents add 6% sales tax. American Laboratory/Labcompare assumes no responsibility for the statements and opinions advanced by the contributors. Change of address: Visit, or provide old mailing label as well as new address, or new address and four-letter, seven-digit subscriber code, in the upper left-hand corner of the mailing label. Include zip or postal code. Allow 46 weeks for address change. POSTMASTER: Send address changes to: American Laboratory, P.O. Box 8613, Lowell, MA 01853-8613. Copyright 2012 by CompareNetworks, Inc. All rights reserved. Reproduction in whole or in part without written permission from American Laboratory/Labcompare is prohibited. ALBYBL 44(2) 148. American Laboratory is registered with the U.S. Patent and Trademark Ofce.

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Editors Page
Attend Pittcon 2012 to Connect With Colleagues, Innovations, and Scientic Discoveries
by Jon N. Peace

ittcon is the worlds largest annual conference and exposition for laboratory science where scientists from all over the world gather to connect with innovations, scientic discoveries, and colleagues. We are looking forward to Pittcon 2012 in the exciting city of Orlando, FL, March 1115, at the Orange County Convention Center. Florida has become the powerful new catalyst in the global life sciences industry. The states biotechnology, pharmaceutical, and medical device sectors continue to grow, making Florida the epicenter of some of the most exciting research, promising discoveries, and successful commercialization efforts in the world. Pittcon will ofcially kick off on Sunday, March 11, with an Opening Session that includes the plenary lecture, Ambient Ionization and Mini Mass Spectrometers: In situ MS for Everyone, presented by R. Graham Cooks, Henry B. Hass Distinguished Professor Analytical Chemistry, Department of Chemistry, Purdue University (West Lafayette, IN). Make plans to attend the complimentary mixer, which will be held immediately following the lecture.

Exposition of Laboratory Instrumentation

The Exposition begins with an Opening Ceremony on Monday, March 12, at 8:30. This years Exposition will offer the latest instrumentation from over 900 leading companies from 40 countries. Recent surveys have revealed that 50% of our exhibitors will launch at least one new product at Pittcon this year. The floor will be maximized for easy navigation and convenient access to amenities such as the tram, Internet cafs, Technology Park, and specialized sections including the Life Science, LIMS, and New Exhibitor areas. The premier Exposition offers conferees a unique opportunity to get a rst-hand look at equipment, participate in live demonstrations, attend informative exhibitor seminars, and meet with technical personnel for Q&A sessions. In addition, 20% of exhibitors will offer discounts on purchases made on site.

tions, and health/wellness. The Technical Program, consisting of symposia, contributed sessions, workshops, and posters, will focus on such topics as environmental, life science, food science, nanotechnology, pharmaceutical, forensics, and alternative fuels just to name a few. Award symposia will recognize and honor scientists who have made outstanding contributions to analytical chemistry and applied spectroscopy. In an effort to help minimize scheduling conicts, there will be no Technical Program during the hours of 11:00 a.m. and 2:00 p.m. daily to allow attendees to visit the exhibits without missing any important technical presentations. In addition, all conferees will have access to webcasts of selected symposia for one year after the conference. The webcasted sessions will be clearly marked in the Final Program. We are very pleased this year to add a new event to our program, a Capstone Lecture, Redesigning DNA: Fixing Gods Mistakes, presented by Steven A. Benner, Distinguished Fellow at the Foundation for Applied Molecular Evolution. This lecture will take place Wednesday, March 14, at 5:00 p.m. in the Valencia Ballroom in the Orange County Convention Center, with a complimentary mixer immediately following.

2012 Technical Program

The 2012 Technical Program is designed to provide a venue for the worlds leading scientists to share their latest research and ndings that impact all areas of laboratory science with an emphasis on worldwide topics critical to current events, opera-

Short Courses
Another educational aspect of Pittcon is the diverse and very affordable Short Course program. Select from over 100 courses to enhance your professional skills in your current area of interest or expand your knowledge of other elds. Our


Short Courses are taught in a classroom setting by instructors who are experts in their elds, and range in length from halfday to two-day sessions. There are 34 new courses offered this year covering areas such as data analysis, food, life sciences, liquid chromatography, management, pharmaceutical, polymers, industrial hygiene, quality compliance, and rheology.

Networking opportunities
Most people would agree that one of the most benecial aspects of attending Pittcon is to take advantage of all the networking opportunities available during conference week. There will be a Twitter caf in the Pittcon booth for those who want to send tweets about Pittcon that will be displayed on a large-screen monitor in Technology Park. For the second year, attendees can get connected to Pittcon before, during, and after the event with the enhanced mobile app, Pittcon 2012, which is available for free download in the App Store and Android Market. Enhanced features will allow you to customize your conference experience by creating your schedule in advance, communicate with other attendees during the event, view exhibitor proles, and take session notes that can be e-mailed. The app will remain available to use as a reference tool after the conference ends. The 2012 Conferee Networking Sessions, which are free to all registered conferees, are a great place to meet people, brainstorm new ideas, and discuss concepts. These two-hour sessions cover a wide range of topics to include forensics, chemical analysis, lab management, environmental, food quality, current trends, and more. Our Employment Bureau helps to connect potential candidates with career opportunities. Interviews can be conducted on-site, and preregistration is required. Postings are suitable for the new entrant to the job market, as well as the seasoned professional. Please visit to stay connected with all that Pittcon 2012 has to offer and learn why thousands of your colleagues attend this premier conference and exposition. Register before February 13, 2012, and pay only $115, which gives you week-long, unlimited access to the Exposition, Technical Program, and Conferee Networking Sessions. We urge you to also take advantage of the lowest prices on hotels by booking through We look forward to seeing you in sunny Orlando for an informative week that is guaranteed to shine! Sincerely,

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by Erica Pike, Michael Rummel, Matthew Trass, Jeff Layne, and Sky Countryman


Fast and Effective LC/MS/MS Analysis of Synthetic Cannabinoids

Table 1

Solid-phase extraction of synthetic cannabinoids from urine using Strata-X-Drug N

Strata-X-Drug N, 100 mg/6 mL 8B-S129-ECH Not required Not required Pretreated urine samples 2 mL 50% methanol in water 5 min under full vacuum 4 mL ethyl acetate:isopropanol (85:15) To dryness at 50 C 500 L 45 % acetonitrile in 5 mM ammonium acetate buffer (inject 2.5 L onto LC/MS/MS)

ynthetic cannabinoids are attracting increasing attention from the drug testing industry because of a sudden surge in the abuse of these compounds. The situation is so severe that an emergency ban of ve synthetic cannabinoids was enforced by the U.S. Drug Enforcement Agency (DEA) in the spring of 2011.1 As a result, toxicology labs are scrambling to develop analytical methods to detect these compounds. This article describes a study designed to develop a method combining streamlined extraction from a urine matrix followed by fast, sensitive LC/MS/MS analysis.24

Cartridge: Part no.: Condition: Equilibrate: Load: Wash: Dry: Elute: Dry down: Reconstitute:

Table 2

LC/MS/MS analysis of synthetic cannabinoids

Column: Dimensions: Part no.: Mobile phase:

Forensic toxicology labs are frequently under extreme time limitations to develop new methods for abused substances when they are identied as critical problems. This time sensitivity was taken into account when developing a solidphase extraction (SPE) method to swiftly analyze the ve synthetic cannabinoids that have been banned by the DEA plus a sixthJWH-122which has also been banned by several states (Figure 1). Synthetic cannabinoids are excreted in urine as glucuronide conjugates that require a pretreatment step prior to SPE cleanup. The pretreatment involves a hydrolysis step to remove the glucuronide from the compounds, enhancing extraction and detection by LC/MS/MS.

Kinetex 2.6 m PFP 50 2.1 mm 00B-4477-AN A: 5 mM ammonium acetate buffer B: acetonitrile Gradient: Time (min) %B 1 45 4 75 5 75 5.01 45 8 45 Flow rate: 0.4 mL/min Temperature: Ambient Detection: API 4000 MS/MS Sample: 1) JWH-200; 2) JWH-200-D5; 3) CP47,497; 4) CP47,497-D11; 5) CP47,497-C8; 6) CP47,497-C8-D7; 7) JCH-073; 8) JWH073-D7; 9) JWH-018; 10) JWH-018-D8; 11) JWH-122

Sample pretreatment
JWH-018 JWH-073 JWH-122

To 2 mL of urine, 1000 L of -glucuronidase solution (containing 5000 F units/mL Patella vulgata in 100 mM acetate buffer, pH 5.0) is added. The mixture is then allowed to hydrolyze for 3 hr at 60 C. The samples are then allowed to cool for 5 min, after which 1000 L of 100 mM phosphate buffer (pH 6.0) is added, verifying that the pH is between 5.5 and 6.5. Samples are then centrifuged for 5 min at 5000 rpm and the pellet is discarded.


CP47, 497

CP47, 497-C8 (Cannabicyclohezanol)

Solid-phase extraction procedure

The Strata-X-Drug N reversed-phase polymeric SPE sorbent was utilized (see Table 1). Strata-X-Drug N differs from other

Figure 1

Synthetic cannabinoids analysis.



Table 3

Absolute recoveries of synthetic cannabinoids from urine

Figure 2

LC/MS/MS chromatogram of extracted synthetic cannabinoids.

SPE sorbents in that it eliminates the condition/equilibration step without sacrificing recovery. The sorbent is also quality control (QC) tested with drugs-of-abuse probes from actual urine samples to ensure that the product performs as expected in reallife situations.

Torrance, CA) coupled to an API 4000 MS/MS (AB SCIEX, Ltd., Foster City, CA). Deuterated standards were also analyzed alongside the extracted compounds to verify proper quantitation and detection (see Table 2).

After analyzing several different wash strengths during the SPE procedure, it was determined that the Strata-X-Drug N sorbent was able to retain the synthetic cannabinoids so tightly that it could undergo a 50% methanol wash without loss of analyte. This produced a clean extract while still providing high recoveries (Table 3). Most reversed-phase SPE methods specify a 510% methanol wash to prevent analyte loss. The elimination of the

LC/MS/MS analysis
After the synthetic cannabinoids were successfully cleaned and concentrated, the eluent was analyzed by LC/MS/MS using a Kinetex 2.6 m PFP core-shell HPLC/UHPLC (ultrahighperformance liquid chromatography) column (Phenomenex,

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Table 4

Polarity switching and multiple reaction monitoring (MRM) transitions

condition/equilibration step also provided time and solvent savings without affecting analyte recovery. The method used to analyze the synthetic cannabinoids was slightly more challenging because it required scheduled polarity switching between ESI (+) and ESI (). Because polarity switching was required, it was essential that there be complete separation between analytes. The Kinetex 2.6 m PFP core-shell HPLC/UHPLC column provided very
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SYNTHETIC CANNABINOIDS continued tion and analysis methods. The work described here demonstrates high-recovery, cost-saving extraction combined with sensitive, reproducible LC/MS/MS for the analysis of six synthetic cannabinoids.

Table 5

Linearity of LC/MS/MS method

1. 2. Health Experts Warn Against Fake Pot, NBC Chicago, June 1, 2010; Chemicals Used in Spice and K2 Type Products Now Under Federal Control and Regulation, United States Drug Enforcement Administration, March 1, 1011; pressrel/pr030111.html. Synthetic Cannabinoids and Spice, European Monitoring Centre for Drugs and Drug Addiction; National Conference of State Legislators; http://www.ncsl. org/?TabId=21398.

good separation of analytes in under 4 min (Figure 2), which allowed polarity switching and time savings (Table 4). Once the LC/MS/MS method was developed, linearity and reproducibility were analyzed by performing six-point calibration curves at varying concentration levels (0.1, 0.5, 2.0, 5.0, 50, and 100 ng/mL). All six synthetic cannabinoids provided correlation coefficient values that equaled 0.999 and above (Table 5), proving that the LC/MS/MS method was not only sensitive but also reproducible.



As the use of synthetic drugs spreads, laboratories must quickly adapt by developing rapid, sensitive, and reproducible extrac-

Erica Pike, M.A., is Sample Preparation Brand Manager, Michael Rummel, B.Sc., is Sample Preparation Product Manager, Matthew Trass, B.Sc., is Application Specialist, Jeff Layne, Ph.D., is Senior Research Scientist, and Sky Countryman, B.Sc., is Manager of PhenoLogix and Applied Technologies, Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501, U.S.A.; tel.: 310-212-0555; e-mail:

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Purchasing Considerations for Field Soil Water Content Prole Probes

ield measurements of soil water content are essential for irrigation managers, consultants, and environmental researchers. Changes in soil water content can infer wettingfront, inltration rate, water-table depth, and root water-extraction zones. For decades, the Neutron Hydroprobe from Campbell Pacific Nuclear (InstroTek, Raleigh, NC) has been considered the industry standard for its accuracy and reliability in making nondestructive, in situ measurements of moisture content along soil profiles (Figure 1). More recently, time domain reflectometry (TDR) and capacitance sensors have been developed to make profile measurements. The Diviner 2000 and
Figure 2 Diviner 2000 with meter, screwcap, and padded cases.

Figure 3


nicantly decrease sampling time (Table 1). Both probes permit data to be stored by the LCD meter, downloaded, or printed from the unit with an RS232 serial cable, but the Diviner 2000s handheld meter enables users to view data graphically in the eld. The EnviroSCAN is the only probe available that is capable of continuous soil water content and salinity measurements from 0 to 40 m deep. With up to 16 capacitance sensors spaced along the field-adjustable probe, repeatability and accuracy are ensured by applying soilspecific calibrations to the sensors individually and using built-in depth and orientation settings. The EnviroSCAN permits real-time continuous monitoring of eld sites, and can be set up as part of an environmental monitoring station where the data are accessed remotely, or by downloading them directly from an individual datalogging device (Table 1). Calibrating these instruments requires obtaining soil samples of varying water contents with a core sampler, calculating the soils volumetric water content, and tting a linear function to the data to estimate calibration coefficients. Recalibration is necessary to check for drift in coefficients at

EnviroSCAN (Sentek Technologies, Stepney, Adelaide, South Australia) capacitance probes are widely used by many researchers and managers. Similar to the Neutron Hydroprobe, the Diviner 2000 (Figure 2) is a portable eld instrument with an LCD digital meter, whereas the EnviroSCAN (Figure 3) is designed to be left in the field for continuous in situ soil water content monitoring and requires an RT6 (Sentek Technologies), CR200 (Campbell Scientific, Logan, UT), or some other datalogging device.

Water content probes

The Neutron Hydroprobe allows for fairly rapid measurements at discrete depths along a 2.44-m prole (minimum, longer cables may be purchased), but the Diviner 2000s automatic depth and orientation sensing of the capacitance sensor (up to 1.6 m) allows swipe-and-go capabilities that sigAMERICAN LABORATORY 13 FEBRUARY 2012

Figure 1 Neutron Hydroprobe on an access tube in the eld.

SOIL WATER CONTENT continued the sensors circuit, the dielectric constant of the soil is determined from the charge time of the capacitor. As soil water content increases, the dielectric constant of the soil increases, which increases charge time along the conductors. The dielectric constants of air (1) and soil (35) are much lower than that of water (80) due to its polarity; therefore, changes in the dielectric constant can be primarily attributed to the changes in water content. Since the soils electrical capacitance changes with soil type, limitations occur in gravelly and coarse stony soils where air spaces are more abundant and the overall capacitance of the soil is low.2

Table 1

Instrument specications and ranges for Neutron Hydroprobe, Diviner 2000, and EnviroSCAN
Neutron Hydroprobe Neutron moderation Yes No 2.44 m standard; longer cable available 060 C 060% Variable 1525 cm 0.5% when calibrated 200 tubes or 2000 readings (24 Kb) 12-V dc rechargeable or 6C-alkaline batteries with ac charger Diviner 2000 Electrical capacitance Yes No 1.6 m in 10-cm increments 070 C Oven dry to saturation 10 cm 0.5% when calibrated 99 tubes Internal battery with ac charger EnviroSCAN Electrical capacitance No Yes 40 m adjustable in 10-cm increments 20 to 75 C Oven dry to saturation 10 cm 0.1% when calibrated Depends on logger 12-V dc solar cell or from logger

Measurement type Field portable Remote access Sampling depth Operating temperature range Moisture range Sphere of inuence Accuracy Memory storage Power

Sphere of inuence and site selection

The sphere of influence, or region of soil measured by the probes, is much smaller for capacitance sensors (10 cm) than for the neutron-emitting source (1525 cm) (Table 1). However, the Neutron Hydroprobes sphere of influence increases as soilmoisture decreases because neutrons have to travel farther before colliding with hydrogen atoms. A large sphere of inuence provides a higher level of sensitivity and accuracy, and a better representation of soil water content, but a smaller sphere size with increased water contents changes the sample size and decreases accuracy in saturated soils. Limitations may be reached in heavy clayey soils with high moisture contents.1 Concessions need to be made when taking shallow measurements because neutrons escaping to the atmosphere from cracks and the soil surface can skew data. Similarly, large cracks read by the capacitance sensor will yield dryer water content readings. Variability in the field is larger for capacitance probes because the sensors are more sensitive to variations in soil macroporosity and soil disturbance during access tube installation due to having a smaller sphere of inuence. The sphere of inuence also affects site selection. For capacitance probes to effectively measure plantwater consumption for irrigation management, the probes must be placed close to the plants or nestled in a row of crops. If placed on the edge of a field, the sphere of influence may be too small to detect root waterextraction.

long-term monitoring and research sites. Additionally, calibration curves will differ between field sites and with sampling tube thickness. The Neutron Hydroprobe, Diviner 2000, and EnviroSCAN all require soil-specic calibrations, but that does not mean that they are all well suited for each soil type and eld setting. The physics must be considered when choosing a probe, because different types of measurements are governed by different principles.

hydrogen from both sources, and the counts will be subsequently higher. Heterogeneity of organic material in the soil profile will be reected in the detector counts and may be explained by considering soil classication and plowing depths. Other chemical constituents, such as boron and chloride, are neutron absorbers and thus affect Hydroprobe readings when concentrations are high. However, readings are not affected by salinity.1 The radioactive neutron emitting source forces users to acquire an Atomic Energy Commission (AEC) permit, which requires that all users be trained in nuclear gauge safety, possess radiation-detecting film badges, and periodically submit the badges to the AEC for radiation monitoring. Costs of registering users on the AEC permits and decommissioning old probes have been increasing; therefore, using the Hydroprobe has become less feasible for many farm and environmental managers.

Neutron emitting probes

The Neutron Hydroprobe contains a 50-millicurie (mCi) radioactive Americium-241/Beryllium source that emits fast neutrons, which collide with hydrogen in soilwater and return low-energy (thermalized) neutrons to the detector. Slow neutrons react with the nuclei of 3He gas in the detector, yielding electrical impulses that can be counted. The count of thermalized neutrons is proportional to the concentration of hydrogen within the sphere of inuence of the radioactive emitter. Since hydrogen is mainly present in soilwater, this becomes a function of water content. However, organic material in soils tends to be acidic and associated with high levels of hydrogen; thus the detector will register

Capacitance probes
The Diviner 2000 and EnviroSCAN rely on capacitance sensors that use frequency domain reflectometry. Since the electrical capacitance of the soil is considered part of AMERICAN LABORATORY 14 FEBRUARY 2012

Proper access tube installation and site location are extremely important to ensure accuracy of the measurements. Improper installation leads to air gaps and preferential ow down the tubes, which has a signicant effect on the capacitance probes. The least amount of soil should be disturbed so that the region being measured is still representative of the surrounding soil. Access tubes may be installed by the standard (i.e., tight-t) or slurry method. Standard installations are recommended, and provide the highest level of reliability because the soil is left intact by using an auger that is slightly smaller than the access tube and securely fitting the tube. The slurry method requires auguring or drilling a larger hole and positioning the tube in a kaolinite and cement slurry. This method is recommended for stony and gravelly soils only because they are difcult to augur, and the risk for air pocket formation is high; moreover, the slurry method is recommended for installations at depths greater than 2.5 m.3 Though it disrupts the surrounding soil that is being measured by the sphere of influence, the moisture content of the slurry equilibrates with that of the surrounding soil. This will create larger problems for capacitance sensors than the Hydroprobe because a greater proportion of the measurement region will be the slurry. The thinner-walled access tubes from Sentek help to compensate for the small sphere of inuence, and can be used interchangeably for the Diviner 2000 and EnviroSCAN. Additionally, the screw-caps that seal the probe into the tube prevent water or particles from getting into the tubes, which maintains accuracy and reliability. This is especially important for the orientation and depth sensing of the Diviner 2000 and EnviroSCAN probes. Neutron Hydroprobe access tubes can be made or purchased from durable 2-in. schedule 40 PVC or aluminum pipe. The increased durability of the material allows the tube to remain in the soil at deep depths for a longer period of time before needing to be replaced.

The Diviner 2000 and EnviroSCAN are less expensive and are not subject to the regulations and permit requirements imposed on the Hydroprobe. However, the Hydroprobe is extremely durable, reliable, and accurate, which is necessary for many research projects.



Hanson, B.R.; Peters, D.W. Soil type affects accuracy of dielectric moisture sensors. California Agriculture 2000, 54(3), 437. Sentek Sensor Technologies. Access tube installation guide for EnviroSCAN, EnviroSMART and Diviner 2000, 2003, Version 1;

1. Kramer, J.H.; Cullen, S.J. et al. Vadose zone monitoring with the neutron moisture probe. Ground Water Monitoring Review 1992, 12(3), 17787. Emily S. Tozzi, M.Sc., is a Research Biologist with SynTech Research, 17915 East Annadale Ave., Sanger, CA 93657, U.S.A., e-mail:

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Life Science

by Jan Prochnow

Automated Powder Dosing in the Life Science Laboratory

reparing analytical samples and standards for the life science laboratory, the pharmaceutical laboratory and the analytical laboratory, is typically a manual procedure that requires that solids be weighed into a volumetric flask and diluted to the mark. METTLER TOLEDO (Greifensee, Switzerland) critically reviewed the work flow and saw an opportunity to automate the sample preparation process while simultaneously reducing the impact of human variability and uncontrolled environmental factors. Specifically, it was predicted and verified that adding liquid by weight improves precision and reduces solvent consumption by 80%. More importantly, weight/ weight sample preparation eliminates the variability of the human element.

it is recommended that the minimum weight be multiplied by a safety factor, typically two or three. Adhering to the minimum weight for weighing the substance ensures that the required accuracy is achieved. Typical concentrations in the pharmaceutical industry require several milliliters of solvent or several grams. Quantifying these amounts on an analytical balance can be done with very high accuracy since this is several orders of magnitude higher than the minimum sample weight, and the uncertainty decreases hyperbolically with the net weight (see Figure 1).

affect the position of the meniscus by wall effects. Weighing both sample and solvents instead of using volumetric flasks improves reproducibility and traceability and minimizes problems associated with volumetric glassware. The vials are small and disposable, which eliminates concern about possible cross-contamination. Hidden costs of washing and sample disposal are also reduced.

Powder dosing
Substance savings
The minimum sample weight and the safety factor depend strongly on the user and environmental inuences. Automated powder dosing with the Quantos powder dosing system (METTLER TOLEDO) within the closed draft shield can thus significantly reduce both the minimum sample weight and the safety factor. For example, according to USP, the XP205 analytical balance (METTLER TOLEDO) has a minimum weight of typically 21 mg and a recommended safety factor of 2. The Quantos automated

Measurement uncertainty
The relative measurement uncertainty of a balance is a hyperbolic function of the weight on the balance (see Figure 1). An upper limit on the relative measurement uncertainty means a lower limit of the weight on the balance. The latter is referred to as minimum sample weight or minimum weight. This minimum weight depends on environmental factors like air movements, stability of the supporting table, and the skills of the user. Therefore,

Sample preparation
The traditional protocol for preparing an analytical sample or standard is to weigh out the specified amount of sample into a volumetric flask on an analytical balance and then dilute with solvent by filling to the mark. Typical volumetric flasks are 25 mL or larger; injection volumes in analytical instruments such as ultrahigh-performance liquid chromatographs (UHPLCs) are only 20 L. Therefore, more than 99.9% of the prepared solution is disposed of without being used. The reason for this excess is twofold: Analytical balances have a characteristic minimum net weight according to USP regulations. Smaller volumetric flasks are not practical because the smaller the flask, the greater the impact of an inaccurate reading of the meniscus. Indeed, when small volumetrics are used, their small size can

Figure 1

Graph demonstrating that uncertainty decreases hyperbolically with net weight.


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POWDER DOSING continued dosing balance has a minimum weight of typically 10 mg and a recommended safety factor of 1.5. This is a significant reduction in the cost of compliance: 65% of substance is saved while still remaining compliant with USP regulations.

Solvent savings
The red curves in Figures 2 and 3 illustrates how the minimum weight, the concentration, and the available flask determine the amount of solvent (Figure 2) and substance (Figure 3) needed. The green curves show the consumption of substance (Figure 3) and solvent (Figure 2) as a function of target concentrations. The red curve in Figure 3 shows that only for four discrete concentrations the minimum net sample weight of 42 mg can be applied. In all other cases, significantly more substance is consumed because one is restricted to quantities of solvent for which glassware exists. In these cases, the

Figure 2 Graph illustrating the solvent consumption as a function of the desired concentration. The red curve applies when solvent is quantied volumetrically using asks, the green curve when weighing the solvent.

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amount of substance needs to be rounded up to the next ask size available. The green curve shows that when the amount of liquid is weighed, for every target concentration only the minimum net weight of substance is required. When quantifying solvent by its weight and not by a volumetric ask, any amount of solvent to match the desired concentration can be used. No rounding up is required. Similarly, Figure 2 illustrates the solvent savings. If a 1.5-mg/mL solution with a 1-g/mL density is prepared manually with a volumetric flask, 50 mL of solvent and 75 mg of substance are consumed. When the solution is prepared automatically and gravimetrically, 10 mL of solvent and 15 mg of substance are sufcient. Substance savings of 80% can be realized. The use of volumetric asks and manual powder weighing results in excessive use of solvent and substance.

Automated gravimetric powder and liquid dosing is very reproducible. To demonstrate this, nine solutions of an active pharmaceutical ingredient (API) were prepared individually, both automatically and manually. To measure the reproducibility, the solutions were analyzed by HPLC. To distinguish between the reproducibility of the sample preparation and the HPLC analysis, 10 repeat injections of the same solution were done. Nine solutions with a target concentration of 0.603 mg/g were prepared. Ten milligrams of API were dispensed automatically into nine 20-mL brown glass vials. Automation allowed 10 mg to be accurately dispensed with an RSD of only 0.89%. Next, the solventa 80:20 acetonitrile:water mixturewas added gravimetrically based on the exact weight of the API dispensed. The RSD of the achieved concentration was 0.001%. The suspension was placed in an ultrasonic bath for 5 min until fully dissolved. Next, a 2-L sample was injected into the HPLC system. The peak areas for the nine individually automatically prepared samples varied with an RSD of 0.19%; the AMERICAN LABORATORY 19 FEBRUARY 2012

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POWDER DOSING continued When a standard preparation is automated by weighing the substance and solvent, the variability in the prepared solutions is insignicant because it is lower than the variability of the analytical instrumentation itself. Automated, gravimetric sample and standard preparation is a paradigm shift that signicantly reduces the cost of each assay and makes the process more reproducible, improving the analytical results.

Until now, the minimum weight of balances and volumetric asks required that 99.9% of prepared samples and solvents were disposed of. Automated and gravimetric sample and standard preparation reduces the minimum weight of the balance and does not depend on volumetric flasks. Therefore, the cost of compliance is significantly reduced. Weight can be determined with very high accuracy, making the process more reproducible and improving the analytical results.
Figure 3 Graph illustrating the substance consumption as a function of the desired concentration. The red curve applies when solvent is quantied volumetrically using asks, the green curve when weighing the solvent.

peak areas for the individually manually prepared samples varied with an RSD of 0.60%. When the same sample was injected 10 times, the peak areas varied with an RSD of 0.21%.

Jan Prochnow, Ph.D. is Head of Product Management and Business Development, Quantos Business Unit, METTLER TOLEDO, Im Langacher, 8606 Greifensee, Switzerland; tel.: +41 449442692; e-mail:

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For One Hundred Years

Food and Beverage

by Mukta M. Shukla

Food Safety Testing and New Technologies

This issue has been brought to the headlines in recent years due to multiple instances of major food pathogen outbreaks resulting in large-scale food poisoning and subsequently massive recalls by food suppliers. Food poisoning is illness related to eating food contaminated by harmful bacteria. According to, the U.S. federal government Web site that provides consumer information related to food safety, one in six Americans is affected by food poisoning each year and the organisms that cause the most illnesses, hospitalizations, and deaths in the United States are: Salmonella, Norovirus, Campylobacter, Toxoplasma, E. coli O157, Listeria, and Clostridium perfringens. While most people recover from food poisoning without any lasting effects, for some, such as older adults, pregnant women, and people with chronic illnesses, the consequences of food poisoning can be deadly or very devastating, including longterm effects such as kidney failure and brain and nerve damage. Thus, it is increasingly important for food producers, suppliers, processors, distributors, and retailers to be aware of the risks and potential food safety issues from the earliest stages of the food production process. This not only saves time and money but also helps improve overall public health.

ust as Alexander Flemings groundbreaking discoveries on penicillin ushered in a new era of antibacterial agents to extend human lifespans, it was the work of Louis Pasteur that helped introduce the food safety industry to new methods to significantly extend product shelf life. Through his work on germ theory, Pasteur (18221895) recognized the presence of microorganisms in food as potential culprits leading to spoilage. He subsequently devised a method for heating food products such as milk and wine to certain temperatures and for specific durations (a process now called pasteurization) to reduce the microorganism burden in such foods. Since those early days, new innovations and technologies have continued to revolutionize the food industry. Nonetheless, with the increasing growth of a complex, global food supply chain, ensuring the safety of food products remains a major challenge; thus, both developed and developing nations continue to confront and address major issues related to food safety.

perishable products designed for very shortterm consumption often by visiting just a single retail location. As I carefully examined the variety of food products available in the U.S. today, I gained renewed respect for the global system that is able to manufacture, transport, store, distribute, and retail them around the world to satisfy consumer needs. I also realized that the challenges of food safety are increasingly complex since the number of players involved in the supply chain of a single food product has increased signicantly in recent years. This, in turn, has increased requirements for implementing various measures for monitoring and quality control at multiple levels, often across national boundaries. In addition, the food industry mostly continues to operate on very thin margins requiring that new technologies be incorporated into the supply chain in a very cost-effective manner. For instance, in one grocery store, I could find whole wheat Indian breads, imported from India, for just $1.99. A friend of mine, who had just returned from New Zealand, was raving about the landscapes and natural beauty of the country, as well as the quality of its dairy products. She was amazed to nd New Zealand cheese at our local grocery store at much lower prices than what she paid there. If either of these suppliers were to increase quality control measures applied to their products, the additional cost of such measures would have to be carefully weighed between being passed onto customers and being absorbed by different players in the supply chain. Such decisions are becoming increasingly important since, while globalization and mass production have significantly increased the quantity, quality, and variety of foods available, they have also introduced new risks such as toxins and new pathogens into the global food system. AMERICAN LABORATORY 22 FEBRUARY 2012

Food safety issues

During this past holiday season, while I was running around grocery stores, buying food and fresh produce to cook meals and entertain family and friends, the fact that I was writing this editorial on food safety was continuously on my mind. As I visited a wide variety of grocery stores from my local Safeway and Giant to Costco, Whole Foods, and Trader Joes, I started to look at the food we consume and its journey from farm to table with renewed curiosity and interest. It is remarkable that Americans (and citizens in many other developed nations) have continuous, year-round access to countless fresh fruits and vegetables from around the world, and that we can purchase such a diverse selection of meat, dairy, and other

Food safety measures

In the U.S. there are a number of agencies that assist in monitoring the safety of food and the health of citizens, such as the Food and Drug Administration (FDA), the Department of Agriculture (USDA), the Centers for Disease Control and Prevention (CDC), the Department of Health and Human Services (HHS), the Environmental Protection Agency (EPA), and the National Institutes of Health (NIH). One of the most significant changes to the U.S. food safety landscape has been the signing of the FDA Food Safety and Modernization Act (FSMA) by President Obama on January 4, 2011. FSMA aims to shift the oversight of U.S. government authorities from management of contamination to prevention of outbreaks.

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FOOD SAFETY TESTING continued Key highlights of the regulation include increased preventative measures such as requirements for more inspections and stringent monitoring. In addition, the legislation enhances oversight of imported foods since, according to the FDA, an estimated 15 percent of the U.S. food supply is imported, including 60 percent of fresh fruits and vegetables and 80 percent of seafood. Lastly, the legislation will also now have mandatory recall authority for all food products, further ensuring that contaminated foods cause minimal damage to public health. With a growing number of participants in the global food market, and with increased oversight and monitoring of the manufacturing and supply processes, the utilization of new technologies, particularly cutting-edge instrumentation such as that used in clinical laboratories, is becoming increasingly important. solutions for pathogen detection. Companies such as Thermo Scientic (Waltham, MA) and 3M (St. Paul, MN) are among major players in culture-based assays. Thermo offers a range of products and services from its Oxoid and Remel brands for microbiological food safety applications. 3Ms products include 3M Petrifilm Plates; 3M Clean-Trace Hygiene Monitoring Systems; 3M Tecra Pathogen and Toxin kits; as well as sample handling, media, and enrichment products. bioMrieux (Marcy lEtoile, France), another leader in the testing eld, offers the VIDAS Immunoassay and VIDAS UP platforms. The UP platform is based on phage recombinant protein assays that provide high sensitivity and stability in detecting specic pathogen strains. time PCR technology. Another new entrant into the molecular space is Roka Bioscience (Warren, NJ), which is launching a new platform based on rRNA targeting to detect pathogens more rapidly and accurately. While these companies represent just a few of the instrumentation providers in the food safety space, the breadth of their technology highlights the ways in which cutting-edge products can be applied to increase preventative measures to monitor the global food supply. Considering that one in six Americans is still affected by food poisoning each year, and that the statistics are likely higher for most developing nations, we will need to diligently, intelligently, and efficiently incorporate new technologies into the food supply to improve not only global human nutrition but also global human health.
Mukta M. Shukla (M.S., Chem., Germany) is co-founder of Glygen Corp. of Columbia, MD, and a Consulting Editor for American Laboratory/Labcompare; e-mail: mshukla@

Food technologies for pathogen detection

Currently there are several players who are leaders in providing food safety technology

Molecular diagnostic techniques

Several companies also provide molecular diagnostics-based approaches. For example, DuPont Qualicon (Wilmington, DE) offers the BAX system, which is based on real-



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Recognizing when an instrument is no longer a cost-effective asset in your lab is relatively easy. Deciding what to do with it next, however, can be a bit more difficult. Would it be better used at another location? Have your needs outgrown its capabilities? And, who will inspect and certify it? These questions and more can be answered quickly and easily by PerkinElmer OneSource. Our asset disposition and redeployment services put a wealth of knowledge, resources and options at your fingertips. By providing real-time visibility into the location and operational status of every instrument through a secure, dedicated online portal, we help you eliminate the inefficiencies associated with labor-intensive audits and replacing lost or misplaced systems. Inventory management time can be cut from days to just hours, freeing you up to focus on other tasks. Efficiencies from the system can result in typical cost savings of 10-15%. This is accomplished through: Accurate asset location and monitoring in real time Protection against over-provisioningmaintain an inventory based on exactly what you need Better laboratory lifecycle management Enhanced asset security Improved asset budgeting Instrument inspection and certification Valuation and verification of quality Complete service from decommissioning to re-commissioning at new location Quick and easy regulatory and financial asset compliance Environmental monitoring to keep instruments running at peak performance

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by Robert Trengove, Bruce Peebles, Katherine Rousetty, Sze Bong, Felician Muntean, Steven Schachterle, Chris Kellog, and Patrick Jeanville

Food and Beverage

sent two key challenges in residue analysis and the drive to improve limits of detection. The use of matrix-matched standards and analyte protectants are two general approaches that have enjoyed significant success, and generally avoid the need for a standard addition approach. Matrixmatched standards have been instrumental in improving the utility of GC-MS by allowing the analysis of certain compounds that have previously been regarded as nonGC compatible. This has provided further condence in identication and quantication of positives when these compounds are screened using both GC- and LC-based analyses. The use of stable isotope dilution, while providing excellent results, represents a very costly and unsustainable alternative for large-scale multiresidue analysis of food.

Pesticide Residue Testing of Grains and Oil Seeds: Minimizing False Positives and False Negatives

C-MS based pesticide residue testing in grains often results in enhanced response, with the degree of enhancement being dependent on the type of grain. Some grain matrices cause matrix interference, leading to false positives and false negatives, as well as signicant limitations in limits of detection (LOD) and quantification, particularly when published multiple reaction monitoring (MRM) assays are used without validation. Matrix-matched standards and analyte protectants are used routinely in pesticide analysis to combat enhancement and suppression, and to provide a more accurate indication of the residue levels present.

uled MRMs with two to four transitions per compound.

Pesticide residue analysis

Analyte degradation and matrix interference with GC-MS based analyses reprea b

Certain pesticide and matrix combinations are well known for illustrating the problems listed above.1,2 It is important to remember that published MRMs should only be used following validation to demonstrate that they are free from matrix interference in the matrix system under study.

GC-MS triple quadrupole

The problem of matrix interference was examined in a variety of grains (wheat, barley, oats, eld peas, chick peas, canola seeds, and soybeans) using the SCION GC-MS triple quadrupole (300 series GC) (Bruker Chemical & Applied Markets Div., CAM) (Fremont, CA). Sample preparation was minimal (2.55 g of grain to compensate for varied lipid content), consisting of a QuEChERS approach. GC-MS conditions were as follows: 1079 inlet with a 5-L injection, Siltek Deactivated Inlet Liner (Restek Corp., Bellefonte, PA), Restek column, transfer line temperature of 270 C, source temperature of 200 C, and schede f

Figure 1 Simazine: 40, 20, 10, 4, 2, and 1 ppb in a) barley matrix overlaid with matrix blank, b) barley matrix-matched standards, c) wheat matrix overlaid with matrix blank, d) wheat matrix-matched standards, e) canola matrix overlaid with matrix blank (S/N for 4 ppb), and f) canola matrix-matched Australian standards (maximum residue limit, MRL, 20 ppb).



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Matrix matching
In this study, GC-MS analysis of whole grain as well as end-of-season grain dust was undertaken in order to demonstrate the necessity for matrix-matched internal standard sets rather than a single internal standard. The purpose behind the dust analysis per se was to determine if it was fit to be used for animal feed or whether it would be used for biofuel processing. This posed a rather challenging problem because the composition of the grain dust was unknown, and there was often a 300400% variation in the internal standard response, depending on the origin of the grain dust samples. An early decision was made to use matrix matching to evaluate matrix interferences on a compound-by-compound and matrixby-matrix approach and elucidate the causes of false positives, false negatives, and compromised LOD. With matrix matching, the authors were put in a better position to determine the limitations of the methodologies and to ascertain the LOD.

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Signal enhancement
With GC-MS, there is usually some beneficial signal enhancement because the matrix protects the pesticides and enhances their transfer from hot vaporizing inlets, reducing thermal stress and masking active sites. Not every compound displays signal enhancement because they are either thermally/chemically stable or have limited adsorption potential. Equally, signal enhancement is not seen with every matrix and some matrices do not provide any signicant protection effect at all. In this study, specic grains contained up to 50% lipids by mass and thus have enormous potential for protection effects. The degree of signal enhancement was dependent on the inlet type. The approach for this study was to deactivate the inlet system to obtain an optimal inert ow path. Various liners were tested, and it was determined that Siltek Deactivated Inlet Liners were the best option for the 1079 inlets. A significant difference in signal enhancement was seen with both the split/splitless and the programmed temperature vaporizer (PTV)-type inlet. Being aware of these variables allowed the false-positives issue to be addressed. AMERICAN LABORATORY 26 FEBRUARY 2012

Figure 2 Malathion matrix interference: 40, 20, and 10 ppb in a) barley matrix overlaid with matrix blank (shoulder from matrix), b) barley matrix, c) barley matrix overlaid with matrix blank, d) barley matrix-matched Australian standards (MRL, 8 ppm), e) wheat matrix overlaid with matrix blank (small interference), and f) wheat matrix-matched Australian standards (MRL, 8 ppm).

Signal loss
False negatives and signal loss are usually caused by differences in matrix types. A good example of this is canola. In this study, canola was also analyzed using chemical ionization (CI), but this did not solve the problem because the signal for one target compound was lost and artifact peaks dominated. The cause of signal loss in this case was probably matrix binding of the compounds.

curve included a concentration range of 401 ppb for the 5-g samples and 802 ppb for the 2.5-g samples. The matrix-matched standards originated from organic wheat, barley, and chick peas, and organic canola


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Matrix interference comparison

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grown specically for this project. Blank matrix solution was generated by using the same QuEChERS protocol used for real samples. All matrix-matched standards were prepared by diluting custom mixtures with matrix solution. Typically, 20 mL of blank matrix was generated at a time and stored in a 80 C freezer until used (shelf life of two weeks once thawed). All the pesticide standards were custom mixes made by ULTRA Scientic (North Kingstown, RI), with seven custom mixtures combined to produce a stable standard mixture.

Simazine matrix interference

Simazine, an herbicide from the triazine class that is used to control broad-leaved weeds and annual grasses, acts by inhibiting photosynthesis; its use has been banned in many countries. Simazine is closely monitored because it is retained in the soil for 27 months following rst application and its entry into water supplies can cause human health problems.3 Figure 1a shows an overlay of the 40, 20, 10, 4, 2, and 1 ppb calibration injections for simazine in barley matrix overlaid with a matrix blank (the at line at the bottom is the matrix blank). There is no signicant interference and the standard curve (Figure 1b) goes down to 1 ppb. The level of detection could be more sensitive and lower levels could be detected, but this gives an example of a situation in which the matrix is not an issue. Wheat shows a similar effect (Figure 1c and d), and again there is a at line for the matrix, showing no interference even at 1 ppb with a S/N of 71; thus, lower detection limits can certainly be achieved.

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Figure 3 Malathion: 80, 40, 20, 4, and 2 ppb in a) canola matrix overlaid with matrix blank (matrix interference at equivalent level 20 ppb), and b) matrix-matched standards.

Canola, with its high lipid content, gives a maximum detection limit of approx. 4 ppb (Figure 1e and f), but this is still well below the Australian Standard MRL of 20 ppb. At 4 ppb with a S/N of 18:1, it could be more sensitive and go a little lower, but can still satisfy the U.S. EPA drinking water requirement.3

malathion 404 ppb trace overlaid with the blank matrix in barley, there is clearly huge potential for matrix interference (Figure 2c). Effectively, the interference level equates to 4 ppb; even if analyte protectants were used, interference would still occur. With malathion in wheat, the interference is much smaller (Figure 2e); the level of detection is down to 2 ppb, as shown on the standard curve (Figure 2f). There is still a very slight interference, but it is not as pronounced as in the barley sample. The interference with respect to malathion in canola does represent a problem, with extracted lipids contributing to the signal at a level of 20 ppb (Figure 3a and b), lowering the effective limit of detection. There is potential to do some cleanup on the QuEChERS extracts to slightly improve this

Malathion matrix interference

Malathion is an organophosphate insecticide of relatively low human toxicity that is used widely in mosquito and boll weevil eradication programs.4 The maximum amount of malathion residue allowed by the U.S. FDA and U.S. EPA on crops used as food is 8 ppm.5 The matrix effects of malathion on various grains were examined using GC-MS techniques. The malathion in barley matrix displayed a slight shoulder from the matrix on the 10-ppb trace (Figure 2a). In the

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problem, but 20 ppb is effectively the best current level of detection.

Phosmet: A challenge in canola

Phosmet is a phthalimide-derived, nonsystemic organophosphate insecticide. Its main application is for controlling coddling moth on apple trees, although it is also used on a variety of other fruit crops. The compound is recognized by many agencies in the U.S. and around the globe as having acute toxicity and human health implications. In barley and wheat matrix, phosmet poses no problem, with little or no matrix interference even at a level of 2 ppb (Figure 4a and b) with a textbook standard curve (Figure 4c and d). The Australian MRL standard for phosmet is 50 ppb; therefore, carrying out this analysis in barley or wheat is not difcult.

Figure 4 Phosmet: 20, 10, 4, and 2 ppb in a) barley matrix overlaid with matrix blank, b) barley matrixmatched Australian standards, c) wheat matrix overlaid with matrix blank (MRL, 50 ppb), and d) wheat matrixmatched Australian standards (MRL, 50 ppb).


Figure 5 Phosmet: a) 80 ppb in canola matrix overlaid with matrix blank (no response), and b) 40 ppb in barley (red = no response) and 80 ppb in canola (green = response).

In contrast, analysis of phosmet in canola does pose a significant challenge. In the trace displayed (Figure 5a), canola demonstrates a massive matrix signal where phosmet should be at 17.489 min. The potential for substantial retention time shift caused by matrix was investigated together with analysis by CI with full scan, yet phosmet could not be detected. This problem is ongoing, and Figure 5b illustrates its extent, with the barley (40 ppb) and canola (80 ppb) traces overlaid (i.e., the canola signal is hidden because of the matrix interference). The obvious answer to this problem is to use LC-MS as an alternative analysis method, but it would be preferable to develop a reliable GC-MS methodology because that technique works very well with every other matrix examined in this study.

The study has illustrated how the matrix can greatly affect GC-MS analysis of some pesticides and how awareness of these limitations can 1) help to establish achievable LOD for each compound and matrix, and 2) minimize false positives when using matrix-specic transitions.

1. DEFRA Report. Development of Methods for the Multi-Residue Analysis of Pesticides in Animal Feeds. aspx?Document=ps2543_9933_FRP.pdf. Cervera, M.I.; Medina, C. et al. Multi-residue determination of 130 multiclass pesticides in fruits and vegetables by gas chromatography coupled to triple quadrupole tandem mass spectrometry. Anal. Bioanal. Chem. Aug 2010, 397(7), 287391. Epub 2010 Mar 17. basicinformation/simazine.cfm. malathion4mosquitoes.htm#malathion. Agency for Toxic Substances and Disease Registry. Public Health Statement for Malathion: phs/phs.asp?id=520&tid=92.


The impact of matrixmatched standards on limits of detection (LOD)

This study has demonstrated that the MRM transitions cannot be utilized in complex matrices without taking matrix interference into account. The authors have tested each matrix one by one and developed a database of reliable MRM transitions on routinely analyzed grain commodities. In some cases, alternative MRM transitions have been necessary to address the issue of false negatives and false positives. The alternative option of using chemical ionization has also been examined, and was found to be extremely useful in certain cases, with enormous potential for future ongoing method development.

3. 4. 5.

Robert Trengove, Bruce Peebles, Katherine Rousetty, and Sze Bong are with the Separation Science & Metabolomics Laboratory, Murdoch University, Murdoch, South St., Western Australia, 6150, Australia; e-mail: R.Trengove@murdoch. Felician Muntean, Steven Schachterle, Chris Kellog, and Patrick Jeanville are with Bruker Chemical & Applied Markets, Fremont, CA, U.S.A.;


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Dionex (Sunnyvale, CA) also recently released a range of SPE cartridges that make it easier and faster to remove contaminants from samples before analyzinghopefully leading to an even clearer chromatogram. The companys ve new types of SolEx SPE cartridges can be added online to an HPLC system to help remove contaminants such as pesticides or pharmaceuticals that can be present at low levels in water. These cartridges are designed for use with the Dionex AutoTrace 280 SPE instrument as well as other SPE instruments.

Optimizing HPLC Sample Preparation

by Caitlin Smith

areful sample preparation is important prior to highperformance liquid chromatography (HPLC)/ultra high-performance liquid chromatography (UHPLC) analysis for at least three reasons. For one, sample concentration often is needed. Second, the sample may require buffer exchange and/or desalting to place it in the appropriate solution for liquid chromatography. Finally, each HPLC or UHPLC sample will need to undergo at least one ltration step to remove contaminants and particles prior to running it through the liquid chromatography column.

Sample preparation techniques

Sample preparation can be achieved using several different methods, and sometimes the best method is dictated by the sample type. However, the most common methods include solid-phase extraction (SPE), centrifugation, and liquidliquid extraction. SPE is one of the simplest and most effective methods, and affordable, disposable SPE cartridges that make the technique very user-friendly are available from a variety of manufacturers (Several newer models of SPE cartridges are described below.) Automated SPE systems also are available from a variety of vendors. Despite the accessibility of user-friendly cartridges and more streamlined protocols for sample preparation, pressure to increase productivity and quality is being felt in the sample preparation eld. As chromatography has become faster and more sensitive, it has put a lot of onus

on getting the sample preparation right, says Vivek Joshi, senior scientist in the technology development group at EMD Millipore (Temecula, CA). In order to get the benefits out of these advances, sample preparation must also become faster and easier to perform. Scientists are more interested in getting their answers and data than spending time preparing samples. The result is many recent advances in methods to increase throughput in sample preparationwhile holding the quality high to keep pace with the increasing demands of UHPLC. Below are some new developments in sample preparation technologies and a few points to consider when choosing products in this developing area.

Importance of ltration in sample prep

Filtering a sample is important for many reasons, foremost among which are clearer chromatographs and cleaner HPLC columns. For HPLC sample prep, the most important thing is particle-free samples, says Joshi. Thats why ltration is a key sampleprep step prior to chromatography. When selecting a filtration device, the following things need to be considered: choice of membrane (based on chemical compatibility with the sample), membrane pore size (based on downstream analysis) and extractables/analyte binding (the lower the better).

SPE cartridges and columns for different sample types

To match the myriad types of samples run through HPLC systems, there are a wide range of tools appropriate to different sample types. For example, Waters (Milford, MA) recently released Ostro Sample Preparation Plate removes multiple families of phospholipids from biological samplesup to 30 times more phospholipids than traditional removal devices or liquidliquid extraction methods. In addition, the companys Oasis SPE products come in several types of chemistries, depending on the sample (for example, different types of SPE sorbents for bases, acids, strong bases and quaternary amines, strong acids, or a mixture of several types). Waters Sep-Pak bonded silica devices also come in a range of chemistries for HPLC and UHPLC preparation, in cartridges, 96-well plates, and Elution plates. AMERICAN LABORATORY 32 FEBRUARY 2012

Syringe lters
Researchers who prepare a smaller number of samples per day (fewer than 10, for example) tend to use syringe lters to lter the samples individually. Scientists preparing high volumes of samples (i.e., more than 100 samples per day) can use multiwell plates and associated robotics to speed up their sample preparation, notes Joshi. But for the majority of users (approximately 65% of scientists) who lter somewhere between 10 and 100 samples per day, neither of these solutions is optimal. Filtering dozens of samples sequentially is laborious, and robotics are prohibitively expensive for medium-volume users.

Vacuum ltration system

EMD Millipore is lling this gap with the Samplicity Filtration System (Figure 1).

HPLC SAMPLE PREPARATION continued This vacuum-based system allows scientists to simultaneously lter up to eight samples directly into standard HPLC vials, says Joshi. The system is unique in that it is a vacuum-based ltration system that relieves the manual force and repetitive-motion stress associated with syringe ltration, one of the most common methods of sample ltration prior to HPLC analysis. More researchers are embracing UHPLC, and this means ltration of the sample prior to injection is more critical, as UHPLC systems are less forgiving when solids are present compared to traditional HPLC systems, says Navin Pathirana, global product manager at GE Healthcare (Piscataway, NJ). Anyone using or switching to such a system should actively consider their ltration step. It is obviously important to choose a lter that functions correctly time after time (for example, without lter bypass) but [researchers] should also consider factors such as ease of use and automation.

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Filtration devices for an HPLC autosampler

GE Healthcares Whatman Mini-UniPrep is designed for an HPLC autosampler, replacing the syringe, syringe filter, vial, septum, and cap. The Mini-UniPrep essentially consists of two parts, an outer chamber and an inner plunger, says Pathirana. The sample is loaded into the outer chamber, and then the plunger is inserted and compressed. The action of compressing the plunger causes the sample to be ltered into the center of the plunger. Once fully compressed, the device can be loaded into the autosampler. Mini-UniPrep is the same size as a standard 2-mL vial. So any autosampler that takes a standard 2 mL vial can take the Mini-UniPrep.

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Filtration System (which filters 24 samples simultaneously) for throughput that falls somewhere between individual samples and 96-well plates.

Automated sample preparation

A wide range of sample-preparation tools are available from Agilent (Palo Alto, CA), which recently released its Agilent 7696 Sample Prep Workbench, a new tool designed for automated sample preparation. The 7696 is unique in that it supports off-line sample preparation for both liquid chromatography and gas chromatography techniques, says Helmut Schulenberg-Schell, marketing manager for liquid chromatography in the Life Science Group at Agilent Figure 1 Samplicity Filtration System from EMD Technologies. In addition, the new Millipore. 1200 Infinity LC Valve Solutions offer a unique exibility to congure for online cleanup and enrichment of analytesunique valving technology amount of residue that accumulates suitable for UHPLC. within the packing of the column, filtering each sample individually is Schulenberg-Schell notes that automainefficient when working with large tion makes sample preparation more numbers of samples. There are numerprecise and more productive, saves ous products available to increase the expensive HPLC-grade solvents, and throughput of sample preparation. For reduces exposure to hazardous reagents. example, for small numbers of samples, Sample preparation is fundamental to Palls (Port Washington, NY) new 0.2-m Acrodisc Syringe Filters were any analytical laboratory, and over half of the work is still done manually, he designed especially to preserve UHPLC says. It is likely that progressively more columns, reducing extractables that can sample preparation will become autoresult in otherwise unexplained peaks mated to some degree, depending on on chromatograms. The companys lthe throughput. Users who run more ters for UHPLC sample preparation are than the occasional HPLC sample may available in several membrane types, have automated sample preparation in depending on the chemistries appropritheir future. ate for the sample. With larger numbers of samples, you might need to bump up to higherthroughput ltration to save time. Several manufacturers offer lter plates that use the common 96-well plate format to lter 96 samples simultaneously. Offerings include Palls AcroPrep 96 Filter Plates. Pall also offers an AcroPrep 24

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Do Your Work Cleanly With Gloveboxes
by James Netterwald

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Glovebox Features Checklist

1 Class 2 Type 3 Application 4 Design 5 Size 6 Safety 7 Material of construction 8 Ergonomics 9 Conguration 10 Customization bator. A pharma manufacturer will want to assemble a drug cocktail within the box to ensure that the drug compound is sterile for use in animals or in humans. Although gloveboxes are primarily unregulated, their sterilization can create an internal standard known as the ISO5. Determined by an international organization for standards, the ISO5 sets the standard for air particle levels inside the box. Once this standard is set, introducing a nonsterile item into the box will destroy the cleanroom standard. If this occurs, only conducting the VHP process again will reestablish the ISO5 standard. In addition to sterility, other conditions inside the box can be controlled. For example, some research and industrial applications require low oxygen concentrations. Very low oxygen requirements inside the box are accommodated by incorporating an oxygen scavenger system, vacuuming out ambient air from the inside of the box, and then backlling the box with a processed gas.

loveboxes are used in laboratories across the sciences. This article will focus on the use of gloveboxes in the life sciencesespecially in those laboratories housed in academic institutions, government institutions, biotechnology companies, and pharmaceutical companies. Purchasers of gloveboxes for these types of settings must consider a number of factors. First, does the application call for isolation (in which case, an isolation box would be used) or containment (i.e., a biosafety cabinet)? In other words, is the purpose of the box to protect a sample against some environmental condition in the external environment (isolation) or to protect operators from a hazard that is inside the box (containment)?

Figure 1 Protector HEPA-ltered glovebox. Courtesy of Labconco Corp.

Gloveboxes as cleanrooms
Because the box is airtight, any samples being worked on within the box are protected from contamination by particulates in the external environment. The internal environment of the box is also important because it creates a specic, clean environment for the work being performed within it. In fact, some types of gloveboxes are also referred to as cleanrooms, isolators, or sterile gloveboxes. Clean refers to the fact that the entire environmentair and surfacesinside the box is sterile, that is, they are free of microbial particulates. The internal environment might also consist of inert gas, low oxygen, a specic temperature or level of humidity, and more. There is no specic glovebox for life science. All kinds of ow boxes can be used in a life science laboratory. The operator should choose the HEPA lter that will work best to remove the size and type of particulate of concern, so that the box will contain clean air internally. Another consideration is whether or not the box should include a processed gas. Often, this means a purged box, which is probably the most common in an isolation application. These boxes are simply purged with a processed gas. For sterile gloveboxes, the most important gas in this category is vaporized hydrogen peroxide (VHP), which is necessary to sterilize the inside of the box.

Applications for gloveboxes

A typical life science application for an isolation box is the preparation of a drug formulation by a pharmaceutical manufacturer. In this application, the manufacturer is looking to sterilize the inside of the box using a validated VHP sterilization process. The United States Food and Drug Administration (FDA) requires that sterilization of the box be conrmed by taking hundreds of swab samples that, when processed, show no growth of any microorganism in an incuAMERICAN LABORATORY 36 FEBRUARY 2012

The isolation box: creating the cleanroom

A lter inside the box handles removal of particulates. There are many choices among filtering systems, the most common of which is the HEPA lter. A HEPA-ltered glovebox, the Protector from Labconco (Kansas City, MO), is shown in Figure 1.

Containment: The class III biosafety cabinet

A class III or biosafety cabinet for life science laboratories is a glovebox for containment of the deadliest organisms known to mankind, for example hanta virus. Those types of organisms are what the U.S. Centers for Disease Control and Prevention (CDC) calls its select agents. For example,

Glovebox design and price points

Gloveboxes can be made of many types of materials, including plastic or stainless steel; the application(s) being performed inside the glovebox determine(s) which material is most appropriate. Box design must accommodate the need for frequent sterilization. Rounded corners can minimize any possibility for cracks or crevices where germs could colonize, thus helping to maintain sterility. Stainless steel allows for easier sterilization than plastic. However, stainless steel boxes are more expensive than plastic ones. Although plastic boxes are less expensive, their durability varies according to the type of material used to fabricate the box. On the higher price end is polypropylene, which resists alcohol and a fairly wide range of harsh chemicals, including biocides. On the lower end is acrylic, which is not as chemically resistant and cannot even be cleaned with alcohol because doing so would eventually cloud and crack the surface. The Series 600 stainless steel glovebox from Terra Universal (Fullerton, CA) is shown in Figure 2. The price point depends on the level of customization. For Terra Universal, a base model of a nitrogen purged box is under $1000. The purchaser can build on the

Glovebox Manufacturers
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Figure 2 Series 600 stainless steel glovebox. Courtesy of Terra Universal.

these boxes can be used to work with smallpox virus, and all the laboratory equipment needed to perform DNA identification of the bug, for example, must be inside this box. These boxes are primarily used in biosafety level IV laboratories and only for nonweaponized organisms. These types of gloveboxes often have a double-door autoclave chamber, and the only way to transfer any equipment or biological materials into or out of the box is through the autoclave.

purged equipment by a having other process control components added to the box. With this model, the user can have a functional system for under $3000. This price point is for the plastic systems. Other companies, such as Labconco, have a multipurpose turnkey type of system with prices starting at $8000$10,000. Stainless steel glove boxes are much more expensive.
James Netterwald, who has a Ph.D. in Microbiology and Molecular Biology and a B.S. in Clinical Laboratory Science, is a freelance biomedical writer and editor; e-mail:

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by Dawn Stickle and Bob Giuffre

Utilizing Extended Linear Velocity to Maximize Peak Capacity in UHPLC

istorically, plate height as of currently available HPLC hardware. given by the Van Deemter With the advent of UHPLC systems, the equation has been used as performance of the columns analyzed can the measure for separation be explored experimentally at these higher efficiency in terms of contribution from power ranges. As such, it would be of interthe A, B, and C term versus linear velocest to test whether chromatographic effiity. Van Deemter theory tells us as you ciency improves at ow rates much higher reduce the particle size of your stationary than typically employed by most chromaphase, the contribution from the A (Eddy tographers. Also, it would be desirable to Diffusion) and C (mass transfer) terms obtain an understanding of how column is reduced and the Van Deemter curve length, column temperature, and gradient is flatter at higher linear velocities. The length perform in terms of chromatographic disadvantage of using smaller particle size efciency as ow rate is increased. is the increase in column backpressure. However, if higher backpressures can be Experimental tolerated by the HPLC, increasing flow rates do not cause a signicant increase in All separations were performed using an Agiband broadening, resulting in faster analylent 1290 Innity UHPLC system (Agilent sis time and higher sample throughput. Technologies, Palo Alto, CA) equipped Recent literature suggests that chromatowith a binary pump (integrated degasser and graphic efciency is improved, though at linear velocities higher than suggested by Van Deemter theory.1 Table 1 Peak capacities for the 100-mm-long column as a function of gradient Column temperature = 30 C 30% max. pressure Gradient time (min) (0.35 mL/min) 5 138 10 211 15 255 Column temperature = 60 C 30% max. pressure Gradient time (min) (0.55 mL/min) 5 145 10 245 15 281 Column temperature = 90 C 30% max. pressure Gradient time (min) (0.8 mL/min) 5 211 10 272 15 320 AMERICAN LABORATORY 38 FEBRUARY 2012

100 L jet weaver gradient mixer), autosampler, thermostatted column compartment, and diode array detector (with lowdispersion 10-mm pathlength optofluidic waveguide ow cell, 1 L dispersive volume). The data were analyzed using a Chemstation from Agilent. Three Agilent Zorbax SB-C18 Rapid Resolution HD columns (2.1 mm diam with 1.8-m particle sizes, stable up to 1200 bar maximum pressure) of varying length (50, 100, and 150 mm), were chosen for the study. The mobile phase was selected as water (A) and acetonitrile (B), both containing 0.1% triuoroacetic acid. The gradient was varied over 5, 10, and 15 min, each time starting at 5%B and ending at 95%B. The column temperature was varied from 30, 60 (tested for 100-mm column only), and 90 C, and the ow rate was varied from 30, 60 (tested for 100-mm column only), and 90% of maximum allowable system backpressure for each condition. The UV signal was monitored at 220 nm (4-nm bandwidth) with a reference of 600 nm (80-nm bandwidth). Peak capacities were calculated for each condition, in triplicate, using a mixture of small molecules (Glafenine, Labetalol, Dipyridamole, Hydrocortisone, Chrysin, and Disperse Yellow, available from Sigma Chemical Co., St. Louis, MO). Peak capacity was calculated by dividing the gradient time by the average

time, percentage of maximum backpressure versus column temperature

Maximizing peak capacity

Although chromatographic efficiency is traditionally described by Van Deemter theory, this theory, in a practical sense, is best used for isocratic and isothermal-based separations. Peak capacity is a more suitable measure of efciency in gradient separation. Peak capacity is dened as the number of peaks that can be separated with dened resolution (for example, a resolution of 1) in a certain period of time for a given system (column length and particle size). Prior to the development of ultrahighpower liquid chromatography (UHPLC) systems, the upper pressure limits and flow rate ranges that could be tested were limited by the power range (flow rate and backpressure operating range)

60% max. pressure (0.75 mL/min) 181 260 308 60% max. pressure (1.15 mL/min) 220 307 352 60% max. pressure (1.6 mL/min) 246 326 379

90% max. pressure (1.2 mL/min) 212 280 324 90% max. pressure (1.8 mL/min) 240 325 365 90% max. pressure (2.3 mL/min) 246 336 387

(most efficient separation) is achieved at 90 C, 90% max. pressure, 15-min gradient, while the lowest peak capacity (least efficient separation) is observed at 30 C, 30% max. pressure, 5-min gradient. Peak capacity increases as ow rate and temperature increase for each gradient time, except for the 5-min gradient time at 90 C, where the peak capacity stays the same as ow rate is increased from 60 to 90% of maximum allowable backpressure. As an example, Figure 1 illustrates the improvement in chromatographic efciency by a comparison of increasing the ow rate from 30 to 90% of maximum allowable backpressure for the 10-min gradient at 30 C. The data were also examined in a different way by calculating the percent increase in peak capacity as the ow rate was increased from both 30 to 60% and 60 to 90% of the maximum allowable backpressure. The results are shown in Table 2. These data indicate that most of the increase in peak capacity is gained as one increases ow from 30 to 60% of the maximum backpressure, although for the most part additional gains in peak capacity are achieved by generating ow rates at 90% of the maximum backpressure. For both the 50- and 150-mm-long columns, peak capacities were also shown to increase as ow rate increased (between 30 and 90% maximum allow backpressure) for each gradient time (5, 10, and 15 min) and temperature (between 30 and 90 C). Additionally, as with the 100-mm-long column, both the 50- and 150-mm-long columns showed their

Figure 1 Chromatography comparison of increasing the ow rate from 30 to 90% of maximum allowable backpressure for the 10-min gradient at 30 C.

peak width (six components averaged over three replicate injections) and adding one. Peak width was taken at half maximum and multiplied by 1.7 (or 4).

Increased peak capacity and chromatographic efciency
Peak capacities calculated for the 2.1 100 mm column as a function of gradient time and percentage of maximum allowable system backpressure versus column temperature are shown in Table 1. The table also includes the flow rates that were used in order to generate 30, 60, and 90% of the allowable system backpressure. The data indicate that the highest peak capacity

Table 2

Peak capacity comparison in terms of percent increase as the ow rate is increased for the 100-mm column
Gradient time (min) 5 10 15 5 10 15 5 10 15 Percent increase (3060% max. ow) 24 19 17 34 20 20 14 17 16 Percent increase (3060% max. ow) 15 7 5 8 6 4 0 3 2

Temperature (C) 30





Table 3

Peak capacity comparison in terms of percent increase as the column temperature increased
Gradient time (min) 5 10 15 5 10 15 5 10 15 Percent increase (30 C) 24 17 12 35 25 21 55 43 38 Percent increase (90 C) 7 3 3 14 19 17 31 20 13

Column length (mm) 50



ity and that increasing the ow rate one can better maximize peak capacity, with much of the peak capacity gain attributed to increasing the gradient time. Additionally, for each column temperature, the longer column has a greater gain in peak capacity as gradient time decreases. The longer the column, the lower the possible ow rate can be at an equivalent percentage of system backpressure. Thus, for longer columns, because one cannot achieve as much peak capacity gains by increasing ow rate, the majority of gain in peak capacity must be achieved using gradient time.


All column lengths showed an increase in peak capacity as flow rate, gradient time, and column temperature increased. Temperature Column Percent increase Percent increase Percent increase For each column length, the largest peak capacity was therefore observed at 90 C (C) length (mm) (5-min gradient) (10-min gradient) (15-min gradient) column temperature, 15-min gradient 30 50 24 17 12 time, at a flow rate generating 90% of 100 35 25 21 the allowable maximum system pres150 55 43 38 sure. Similarly, for each column length, 90 50 7 3 3 the smallest peak capacity was therefore observed at 30 C column temperature, 100 14 19 17 5-min gradient time, at a ow rate gen150 31 20 13 erating 30% of the allowable maximum system pressure. In order to increase the highest peak capacity at 90 C, 90% max. the column, the lower the possible ow rate peak capacity of a separation, the column pressure, 15-min gradient, while the lowest can be at an equivalent percentage of system temperature can be increased and/or the peak capacity was observed at 30 C, 30% backpressure. Therefore, for longer columns, gradient made shallower (longer run times), max. pressure, 5-min gradient. because one cannot achieve as many peak but the maximum peak capacity is achieved capacity gains by increasing flow rate, the by also increasing the flow rate. If higher The gains in peak capacity (as a function of majority of gain in peak capacity must be throughput is needed and shallow gradients increasing flow rate) by changing column achieved using column temperature. cannot be used because of their longer run temperature are shown in Table 3. The gain times, ow rate should be increased to greatly is expressed in terms of the percent increase Similar to the discussion about column temimprove (but not maximize) peak capacity. in peak capacity as the flow was increased perature, the gain in peak capacity (as a funcAlso, if a particular column or analyte canfrom 30 to 90% of the maximum system tion of increasing ow rate) by increasing the not tolerate high column temperatures, ow backpressure. For each column length and gradient time can be compared, as shown in rate can be increased to greatly improve (but gradient time, the percentage increases are Table 4. Again, the gain is expressed in terms not maximize) peak capacity. largest at lower column temperatures. Since of the percent increase in peak capacity as the percent increase in peak capacity at 90 the ow was increased from 30 to 90% of the Reference C is not as signicant as at 30 C, this can maximum system backpressure. For each col1. Petersson, P. J. Sep. Sci. 2008, 31, be interpreted to mean that for a given column length and temperature, the shorter gra234657. umn and gradient time, the higher the coldient resulted in greater percentage increases umn temperature, the higher peak capacities, in peak capacity, aside from increasing the and that increasing the ow rate one can betgradient time from 10 to 15 min on the Dawn Stickle, Ph.D., is LC/MS Application Sciter maximize peak capacity, although most 50-mm column, where it stayed equal. Since entist, Agilent Technologies, 2850 Centerville the percent increase in peak capacity at lonof the peak capacity gain is achieved using Rd., Wilmington, DE 19808, U.S.A.; tel.: 302ger gradient times becomes less signicant, increasing column temperature. Addition636-3510; e-mail: this can be interpreted to mean that for a ally, for a given gradient time, column temBob Giuffre is LC Application Scientist, Agilent given column length and temperature, the perature has a greater gain in peak capacity longer the gradient, the higher peak capacas the column length increases. The longer Technologies, Budd Lake, NJ, U.S.A.

Table 4

Peak capacity comparison in terms of percent increase as the gradient length increased


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Conference Review

by Robert L. Stevenson

Highlights and Challenges in Laboratory Automation: Review of the 4th Annual Automation User Group Meeting

In 2006, throughput was increased to 2.8 TB/year. By 2010, throughput increased still further, to 50 TB/year, and in August 2011, the rate was 500 TB/year and accelerating. This involves 51 systems (Agilent liquid handlers feeding the HiSeq 2000 [Illumina Inc., San Diego, CA]) that automatically analyze about 10,000 samples per month.1 For the most recent round of instrumentation, Agilent was selected as the vendor for the sample prep stage, which uses Agilent Sure Select Enrichment chemistry implemented on its Bravo automation platform. Ms. Fisher advised that full automation improves consistency and reduces contamination. No touch is the current criteria to reduce contamination. Acoustic liquid handlers from Labcyte (Sunnyvale, CA) are used to decrease plastic waste burden with air-driven pipetting. Fisher pointed out that automation generally does not mean a reduction in fulltime employees. However, when the automation is successful, one has the data and can then focus on improving the process even further, such as throughput, resolution, and data quality, or move on to distill knowledge from the data.

or four years now, Agilent Technologies (Sunnyvale, CA) has hosted an annual users conference on laboratory automation. The 4th Automation User Group Meeting was held at the Mark Hopkins Hotel in San Francisco, CA, from September 20 to 23, 2011. Key points of reports are: Automation of sample prep increases data quality and quantity while reducing costs, and laboratory productivity largely depends on design of experiments (DoE) and work flow. Todays automated systems for chemical and biochemical assays, such as the Agilent BioCel, integrate modules and software from many vendors. Proposed next-generation systems should share a common industrial design for the modules to improve layout and servicing.

with t to purpose. But their business is also global: Amgen wants the service level to be high and globally uniform. Automation facilitates standardization of lab protocols across functions and along the R&D maturation path. For instance, as a small-molecule product candidate goes through research, preclinical, and development phases, the synthetic processes are scaled up to meet the needs of each phase. Scaleup means change. Grandsard pointed out that companies are working on unit synthetic processes that can be the same from the lab to the factory oor. The latter would just use many more of the units running in parallel. The example he gave was synthetic chemistry modules that can scale from mg/unit to kg/room by using the required number of units running in parallel. This approach might reduce concern that scaleup of products might change the product from that which was used in the original NDA.

Sample prep and automated liquid handling

Scott Fulton of BioSystem Development (Madison, WI) lectured on the AssayMAP Microchromatography Platform for sample prep using automated liquid handlers such as the Bravo. Disposable plastic cartridges contain 5 L of sorbent constrained by upper and lower support lters. As with solid-phase extraction, the particular sorbent can be selected from a variety of surface chemistries bonded to 15100 m particles. Using a special 96-well head, the Bravo precisely controls, aspirates, and dispenses with ow lower than 1 L/min. Pmax is 20 bar. Fulton explained that a low liquid ow is essential in quantitative extraction. Mark Novotny of the J. Craig Venter Institute (San Diego, CA) pointed out that low ow rate dispensing nearly eliminated interwell contamination in his sample prep of DNA from single bacteria. According to Hinnerk Boriss (Sovicell, Leipzig, Germany), estimating the unbound fraction of drug candidates is essential for

Laboratory automation: Focus on the biopharmaceutical industry

The meeting opened with an economic analysis of laboratory automation, with a particular focus on the biopharmaceutical industry, given by Dr. Peter Grandsard of Amgen (Thousand Oaks, CA). Biopharm is growing, especially since many new drug candidates are constructs of large and small molecules. Both regulators and developers demand a sciencedriven approach for development. He separated R&D efciency from effectiveness, where efciency measures the cost and time to introduction. Big pharma, which includes Amgen, suffers from low productivity, as measured by launched products. Thus, instrument purchases are under cost-cutting pressure. The phrase top of the line is being replaced

Parallel systems
Massively parallel instruments for research seem to work. The Broad Institute in Cambridge, MA seeks to develop and apply systematic approaches in the biological sciences to dramatically accelerate the understanding and treatment of disease. The evolution of genome sequencing at Broad was described by Ms. Sheila Fisher. Broad was one of the leaders in sequencing the human genome and follow-on genomes. In 2005, the Institute operated 117 ABI 3730 DNA sequencers (Applied Biosystems/Life Technologies Corp., Carlsbad, CA). Productivity was about 60 GB/year, which is about one genome, since high redundancy is needed. AMERICAN LABORATORY 42 FEBRUARY 2012

predicting their pharmacodynamics. Sovicell developed the TRANSIL brain absorption assay for measuring the transport across a porcine lipid by layer. The assay correlates well with conventional dialysis methods, but is much faster, requiring less than 30 min on the Bravo liquid handler. Final quantitation is with RapidFire TOF-MS (also from Agilent) or with LC-MS, which is slower. Sovicell offers assays for liver binding as well. Peptide samples can also be prepared with the Bravo liquid handler using PepTips from Glygen Corp. (Columbia, MD) in a 384-well format. Peptides (1520 mers) are synthesized in microtips using FMOC chemistry. Run time is less than 24 hr. The peptides can be used in the tip or eluted into a 384-well plate for further use.

The next generation of laboratory automation

Looking at the state-of-the-art, most of the workstations (such as the BioCel) seem to be a collection of components with a robot as the frenzied orchestra conductor. Each piece of apparatus looks like a kluge. Some units accept plates in the portrait mode, and others in landscape mode. Some have top entry and exit, with others on the side. Real estate around the robot is scarce and often difcult to access. The industrial design for next-generation instruments should be looked at from an industry-wide perspective. Clearly there is room for improvement in current design: Modules could form a circle surrounding the robot, which would have an industrystandard inner diameter. Each module would receive and expel the sample plate in portrait format. The rear would provide access for loading and servicing. Heights could be quantized also, with 25, 50, or 75 cm to promote stackability. Widths could be 60o to accommodate six modules/layer. Robots should be extendable from nearoor to near-ceiling heights. Waste processing needs attention also. Agilent is to be congratulated for organizing a balanced and informative program that included both chemistry and instrumentation. Agilents market focus is midmarket labs that run experiments of about 10,000 wells or smaller. This segment should grow rapidly, since many assays are being developed, which will increase applications. Also, funding should be more readily available for systems in the $500K to $5 million range. This is in contrast to ultrahigh-throughput screening systems that usually reside in core facilities serving the enterprise, often running experiments with a million wells.

Integrated automation solutions and work ow organization

Laboratory automation instrumentation is a fragmented market. A few vendors, such as Beckman (Fullerton, CA), offer a limited number of workstations that focus on particular applications. Generally, the major vendors offer freestanding modules. Even a modestly complex work ow will usually involve components from two or more vendors. Who integrates all of these components into a system? Some users might be adept at interconnecting the mechanical layout to comply with the work flow, but few would list software integration of instrumentation as a core competency. In practice, there are no small software problems. Writing drivers to interface from the system controller to the modules requires cooperation of the various vendors, plus technical competence in programming. In prior years, Agilent and H-P had minimal involvement in systems integration or special instruments. For example, in GC, requests for specials were routed to the Channel Partner program. The BioCel program from Agilent is designed to design, sell, assemble, and support custom-automated systems for chemistry and biochemistry. Agilent supports over 600 modules from nearly 100 vendors, providing mechanical and software integration via VWorks software.

$9,950 $11,995 $14,995 $30,500

$10,500 $10,795 $14,600 $5,995

1. zhtml?c=121127&p=irolnewsArticle&ID=1435009&highlight; accessed Oct 7, 2011.

Robert L. Stevenson, Ph.D., is a Consultant and Editor of Separation Science for American Laboratory/ Labcompare; e-mail:


Statistics in Analytical Chemistry

by David Coleman and Lynn Vanatta

Part 46R (Concluded)

Fortunately, there is a statistic known as R2adj (where adj stands for adjusted); its value gives a more honest assessment of the models adequacy. Below are the details. In a sentence, R2adj includes a penalty for each term used in the regression. If the additional term(s) is not needed, R 2adj will generally decline relative to its value for the previous (simpler) model. Mathematically, R2adj is a modication of Eq. (2) above; the new formula includes degrees-of-freedom (DOF) terms: R2adj = 1 (MSError/MSTotal) where: MSError = Mean Square Error = SSError/DOFE DOFE = degrees of freedom for Mean Square Error MSTotal = Mean Square Total = SSTotal/DOFT DOFT = degrees of freedom for Mean Square Total Two questions jump to mind. First, how are the DOF terms calculated? Second, why is Mean used to describe the adjusted Square terms? The answers are as follows. In general, DOF terms for a statistic are computed by starting with the number of data points that are in the data set under discussion. Every time a calculation is made using this original set, a degree of freedom is lost for any statistic that depends on the calculation. SSTotal is calculated using the entire set of raw responses; the total number of data points in a set is typically designated as n. However, to calculate SSTotal, one must rst calculate the average of all the responses, thereby sacricing a degree of freedom. (See Part 44 or Part 45 for the formulas for the SS terms.) Thus, the associated DOF term is (n-1). For SSError, the starting point is the same as above. However, this time, a model must rst be tted to the data, since the predicted responses are needed in the calculation of this statistic. Each parameter (p) in a model includes a coefficient, which must be calculated; for example, a straight-line model requires the calculation of an intercept, as well as a coefcient for the x term, so a degree of freedom is lost for each parameter. As a result, the general expression for DOFE is (n-p). The use of Mean Square to describe the terms in R2adj can be understood by thinking about what happens when one calculates the mean (i.e., average) of a set of data. The formula is the sum of all the values, divided by the total number of data points. In other words, the sum is divided by the degrees of freedom. In this case, no degrees of freedom were lost beforehand, since this determination is based solely on the original data. Thus, n is the appropriate DOF value. Since MSError and MSTotal also divide a sum by the associated DOF term, the use of Mean in the names is logical. The stage is now set for deriving a more useful formula for R2adj. (3)

ver the course of the past two articles (Part 44, Oct 2011, and Part 45, Nov/Dec 2011), R 2 has been dened, its components have been explained verbally and mathematically, and the statistics formula has been presented. Also included has been a discussion of the limitations of R2, and the traps that lie in wait for those who rely exclusively or too heavily on this number. Is there a way to cast this often-used statistic in a more favorable light? This installment will address that question, as well as offer a more robust path for evaluating candidate models. To review the bidding, the formula for R2 is: R2 = SSModel/SSTotal, or R2 = 1 (SSError/SSTotal) (1) (2)

Recall, though, that SSError includes the random noise inherent in the data, as well as the variation the model fails to capture. Furthermore, the value of R2 will increase (or remain unchanged) every time another term is added to the model. As was explained earlier, these two facts can lead the user down the primrose path.


Incorporating the two DOF expressions into Eq. (3) results in the following: R2adj = 1 [SSError/(n-p)]/[SSTotal/(n-1)] (4) Regrouping yields: R2adj = 1 [(SSError/SSTotal)] * [(n-1) / (n-p)] (5) Rearranging Eq. (2) gives: SSError/SSTotal = 1 R2 Combining Eqs. (5) and (6) yields: R2adj = 1 {(1-R2) * [(n-1)/(n-p)]} (7) In Eq. (7), the last expression, [(n-1)/ (n-p)], can be considered a penalty factor that keeps R 2 adj honest. In other words, the inclusion of DOF terms levels the playing eld somewhat when different models are compared using R2adj. Keep in mind, though, that even R2adj must be used with caution. Recall the example in Part 45. There, a data set was tted with four different models: 1) quadratic, 2) cubic, 3) quartic, and 4) quadratic + phases-of-the-moon (POM) term. The values for R2adj stack up as follows: Quadratic Cubic Quartic Quadratic + POM 0.8735 0.8688 0.8652 0.8703 (6)

uation should be on the tools of: 1) the p-value for any term that was just added and 2) the residual plot and the related lack-of-t (LOF) test. (For details related to this alternative, see Parts 9, 10, 22, and 23 of this seriesAmerican Laboratory, Feb 2004, Mar 2004, Jun/Jul 2006, and Oct 2006, respectively.) First, if the p-value of the new term is insignicant (i.e., >0.01), then the term is not needed and its inclusion will result in overfitting. In the case of a straight line, the x-term is the lines slope, which will typically increase in a plot of raw responses versus concentration, thereby being significant unless there is a major problem with the instrument. Second, the residuals pattern will help the user determine if the model exhibits lack of fit; random scatter about the zero line suggests an adequate model. The LOF diagnostic is based on the residual values and does separate SS Error into its parts. Thus, the door is open for distinguishing between random noise and leftovers from the model, and for producing a p-value that will reect the sufciency of the chosen curve. The usefulness of this alternative approach can be shown by returning to the moon example. In Part 45, the scatterplot displayed curvature, so a quadratic was selected as the rst candidate. The wisdom of this decision can be seen in the results of the LOF test; the p-values for a straight line and a quadratic were 0.0276 and 0.9363, respectively. The residual patterns in Figure 1 agree with the LOF test. Furthermore, the p-value for the quadratic term is 0.0008, indicating that x 2 is needed in the model. Addition of either an x 3 or POM term results in insignificant p-values for the new member (0.8935 and 0.5729, respectively). Thus, there is conrmation that a quadratic model is adequate; inclusion of additional terms will result in overtting. One additional matter is the choice of tting technique (see Part 8, Nov 2003, for details on this topic). Neither R2 nor R2adj can help here, either. The proper way to AMERICAN LABORATORY 45 FEBRUARY 2012

The progression from the rst through the third models is accompanied by a decrease in R2adj, thereby signaling the inclusion of inappropriate terms. (Note that this comparison is for illustrative purposes; one is splitting hairs by looking at essentially the third decimal place of R2adj.) Comparison of the POM-containing option with the cubic might lead the casual observer to think that he or she was getting somewhere, and that connecting a cubic with the moon might lead to victory! It is time to turn to a more reliable (although more complex) alternative to either R2 or R2adj. The focus in model eval-


Figure 1

Residuals plots for a simulated data set t with a) a straight-line and b) a quadratic model. See text for details.

evaluate ordinary least squares versus weighted least squares is to model the standard deviation of the responses; if there is trending with concentration, then the latter technique is needed. The nal take-home message is that even R2adj is not a strong tool for evaluating model selection or tting-technique choice, and should be employed only when accompanied by a very

large grain of salt. Instead, users should depend on residual patterns and the LOF test, and standard-deviation modeling, respectively. The authors cannot overemphasize the importance of these two emboldened statements!
David Coleman is an Applied Statistician, and Lynn Vanatta is an Analytical Chemist; e-mail:

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Raman Instruments for Materials Verication Light Scattering Detector


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B&W Tek, Inc. offers The miniDAWN TREOS from Wyatt Technology Corp. is a NanoRam handheld instrucompact, simple-to-use multiangle light scattering (MALS) detector ments for materials identicafor HPLC. It connects quickly to existing chromatography equiption and verication within ment and allows users to determine the absolute molar masses and cGMP-compliant facilities. The sizes of polymers or biopolymers without the need for reference stanstate-of-the-art compact Raman dards or column calibration. The patented, multiangle detector array spectrometer and integrated works for molecules of just a few hundred daltons to several million. computing system is based on an open systems architecture design that can support a broad range of applications in multiple industries. Designed for use by nonspecialists, it is easy to use and operates single-handedly. The instruments allow rapid development of standardized and validated Company Page URL ACHEMA 2012 ...................................................................................46 methods to facilitate 100% inspection for purity AirClean Systems ............................................................................21 and quality, as mandated by government regulators Anton Paar .........................................................................................11 within operational guidelines provided by U.S. Applied Scientic Instrumentation ..........................................24 ...................................................... and international pharmacopeia. Aqua Solutions, Inc.........................................................................12 ............................................................... BrandTech Scientic, Inc. ............................................................35 .......................................................

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The January cover image showed a lymph node, specically, a 250m projection into a mouse popliteal lymph node where immune cells (red and yellow) congregate; imaged with multiphoton microscopy. Collagen bers (white and purple) forming the lymph node capsule were visualized with second harmonic generation. The lymph node was excised from a mouse. Red and green dyes were used to delineate two different populations of immune cells. The specimen was immersed in PBS and imaged with a 10 water immersion lens. Has this months vibrant image captured your imagination? Add your comments and see what other readers are saying at If you have a thought-provoking image, please submit it to

Brookeld..........................................................................................19 ................................ Buck Scientic ................................................................................43 Carver, Inc. .......................................................................................20 .................................................... Centerchem, Inc. ............................................................................20 .................................................... Coy Lab Products ........................................................................... 37 Drummond Scientic Co. ....................................................... 18, 23 Environmental Express ................................................................28 ............................................................. Eppendorf North America, Inc.....................................................17 ....................................................... Erlab Inc. ............................................................................................10 Fluid Metering, Inc. ..........................................................................11 .......................................................... Gilson.................................................................................................47 Glas-Col .............................................................................................26 ....................................................... Hanna instruments, Inc. ...................................................Cover 4 Hellma Analytics ............................................................................34 JULABO USA, Inc. .............................................................................41 ............................................................... KNF LAB .............................................................................................26 ............................................................... Labnet International, Inc. ............................................................45 Leco Corp. ............................................................................27, 29, 31 ................................................................... MicroSolv Technology Corp. .......................................................47 NuAire, Inc.......................................................................................... 3 Omega Engineering, Inc. ................................................................. 1 .............................................................. Omni International, Inc. ...............................................................33 .......................................................... Peak Scientic .................................................................................. 7 ................................................ PerkinElmer, Inc. ..................................................................... 24A-D PLAS-LABS, Inc. ...............................................................................47 Poly-Pipets, Inc. ..............................................................................34 Postnova Analytics........................................................................30 ......................................................... PRO Scientic Inc. ...........................................................................19 Quadrex Corp. ................................................................................. 27 Sartorius Corp. ................................................................................. 5 Scientic Industries, Inc. .............................................................44 ...................................... Shimadzu Scientic Instruments Inc. ............................Cover 2 Sonntek.............................................................................................48 Terra Universal .................................................. With Some Issues Tiger Optics .....................................................................................28 ...................................................... UVP .....................................................................................................24 .................................................................... W.A. Hammond DRIERITE Co., Ltd...............................................39 Wako Chemicals USA, Inc. ............................................................47 ........................................................ Wasson-ECE Instrumentation ....................................................... 8 ............................................... Wyatt Technology Corp. .....................................................Cover 3 ................................................................ Xylem Inc. ..........................................................................................15 ................................................................... Find us on Follow us on



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